CNS Repopulation by Hematopoietic-Derived Microglia-Like Cells Corrects Progranulin deficiency

Hematopoietic stem cell transplantation can deliver therapeutic proteins to the CNS through donor-derived hematopoietic cells that become microglia-like cells. However, using standard conditioning approaches, hematopoietic stem cell transplantation is currently limited by low and slow engraftment of microglia-like cells. We report an efficient conditioning regimen based on Busulfan and a six-day course of microglia depletion using the colony-stimulating factor receptor 1 inhibitor PLX3397. Combining Busulfan-myeloablation and transient microglia depletion results in robust, rapid, and persistent microglia replacement by bone marrow-derived microglia-like cells throughout the CNS. Adding PLX3397 does not affect neurobehavior or has adverse effects on hematopoietic reconstitution. Through single-cell RNA sequencing and high-dimensional CyTOF mass cytometry, we show that microglia-like cells are a heterogeneous population and describe six distinct subpopulations. Though most bone-marrow-derived microglia-like cells can be classified as homeostatic microglia, their gene signature is a hybrid of homeostatic/embryonic microglia and border associated-macrophages. Busulfan-myeloablation and transient microglia depletion induce specific cytokines in the brain, ultimately combining myeloid proliferative and chemo-attractive signals that act locally to repopulate microglia from outside the niche. Importantly, this conditioning approach demonstrates therapeutic efficacy in a mouse model of GRN deficiency. Transplanting wild-type bone marrow into Grn−/− mice conditioned with Busulfan plus PLX3397 results in high engraftment of microglia-like cells in the brain and retina, restoring GRN levels and normalizing lipid metabolism.

improve and reduce the potential effects of CSF1Ri on hematopoietic reconstitution and 124 intervention time, we tested the effectiveness of administering PLX before transplantation.

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Histological analysis showed a widespread and homogeneous distribution of BM-derived GFP+ 147 cells throughout the brain, spinal cord, and retina in mice treated with BU + PLX (Fig 1g-

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Therefore, achieving fast repopulation of the CNS by hematopoietic cells would significantly 154 improve HSCT's efficacy for neurological diseases with rapid progression. To examine the kinetics of brain repopulation, we looked at freshly isolated microglia preparations at 1, 4, 8, and 20 days 156 after PLX withdrawal (corresponding to 21-, 24-, 28-, and 40-days post-transplantation, Fig. 1i).

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Flow cytometry analyses showed a dramatically depleted microglia compartment followed by fast 158 repopulation by GFP+ CD45+CD11b+ cells that peaked after 20 days (Day 40 post-transplant, 159 Fig. 1j-k, Supplementary Fig. 1c). BU administration alone did not significantly reduce the

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Previous studies on cytokine secretion in the conditioned brain have focused on pro-inflammatory 172 cytokines such as TNF-a IL1-b, IL1-a, and known myeloid chemokines such as CCL2 (also known 173 as monocyte chemoattractant protein 1, MCP-1), CCL5, and CXCL10 11,17,60 . These studies 174 consistently show that irradiation stimulates more pro-inflammatory cytokines than Busulfan. To 175 elucidate mechanisms of recruitment and repopulation in our combined regimen, we measured a 176 panel of 50 cytokines in the brain of BU + PLX-treated mice and compared them to untreated 177 mice (U). Cytokine quantification at 1, 4, and 8 days after PLX3397 withdrawal showed 178 unchanged levels of pro-inflammatory cytokines such as IL1-b, IL-1a, IL6, and TNF-a (Fig. 1l).

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were also transiently elevated, which is consistent with their role in promoting the mobilization of 184 myeloid cells (Fig. 1n-o and Extended data Fig. 4a). Interestingly, SDF-1 (also known as 185 CXCL12), a potent chemoattractant for hematopoietic cells 61 , was also increased the brain (Fig.   186 1n). Most cytokines, except IL34, BAFF, and CXCL10, returned to baseline by day 8 (Fig. 1m   187 and Extended data Fig. 4a). The induction of CCL11, CCL7, and CXCL10 in the brain was not paralleled by increases in other pro-inflammatory cytokines normally co-induced during immune-189 derived inflammatory processes 62,63 (Fig. 1l, o and Extended data Fig. 4a). Notably, the cytokine 190 elevations following PLX withdrawal were brain-specific and were not detected in plasma

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We also examined the reconstitution of tissue macrophages in the heart, liver, lung, and 217 peritoneum. Long-term engraftment of donor-derived GFP+ macrophages (MF) in these tissues 218 had similar efficiencies in BU + PLX-and BU-treated mice (Fig. 2 i-j,). Kinetic analysis of the 219 depletion and repopulation of peritoneal macrophages in BU + PLX-treated mice showed   engraftment of donor-derived macrophages (GFP+ CD45+ CD11b high ) in the peritoneum of adult C57BL/6 mice before and after BU + PLX as measured by flow cytometry. The experimental timeline is depicted in Fig. 1i. n=3 mice/cohort; * vs. Untreated (U). l-o, Behavioral analyses performed between 6 and 7 months post-BMT in mice analyzed in Fig. 2 a-j; BU n=10, BU + PLX n=10, Untreated n=10. l, spontaneous locomotion analyzed using the Activity chamber test; the total distance moved (periphery plus center) is reported. m, exploratory behavior analyzed using the Activity chamber test; the total vertical counts (periphery +center) are reported. n, spatial memory analyzed using the three-arm Y-maze test; the percentage of spontaneous alternation across the arms is reported. o, recognition memory analyzed using the Novel object recognition test. The percent time spent interacting with the Novel object (Object 3) and the time spent interacting with previously encountered objects (Object 1 and 2) are reported. a-i, k-o, Data are reported as Mean ± SD. Statistical analysis: a, c, e, g, two-way ANOVA with Tukey post-hoc; b, d, f, h, i, multiple t-test with Holm-Sidak correction; k, oneway ANOVA vs. Untreated with Dunnett post-hoc; l-o, one-way ANOVA Tukey post-hoc.
complete MF depletion at the time the drug was removed (day 21) and complete repopulation 221 three weeks after (day 40, Fig. 2k and Supplementary Fig. 2b). Although no differences were 222 found in the long-term chimerism in tissue macrophages, PLX accelerated macrophage 223 replacement whether administered pre-or post-transplant (Extended Data Fig. 4i-j).    showing the differential expression of microglia signature genes in the brain clusters depicted in panels c-d.
cluster and cluster overlap is depicted in Fig. 3d

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To u n d e r s t a n d a n d d e f i n e m i c r o g l i a a n d MGLC transcriptional states, we examined the expression 263 of signature genes reported across multiple studies (Supplementary Table 1 Overall, based on the expression of well-defined homeostatic microglia genes, the 286 fraction of "homeostatic" cells within the MGLC population was estimated to be 83% which was 287 lower than the 99% estimated for host MG ( Fig. 3d-g). In addition to the combined analysis, we 288 also performed differential gene expression analyses and evaluated the single-cell heterogeneity

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The CD11b+ population in the brain also includes border-associated macrophages (

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We next evaluated signature proteins expressed in BAMs, microglia, hematopoietic, and Mean expression in group Brain-associated macrophages (BAM)

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We found a few DAM genes upregulated in MGLCs compared to naïve MG (Fig. 5a). ApoE and 340 Lyz2, were highly expressed in most MGLC clusters and were also upregulated in conditioned 341 host MG (Fig. 5a). Other DAM genes found to be increased in MGLC clusters were Axl, Ifi27l2a, 342 Gpr65 and Igf1. MGLCs did not express many other well-known DAM genes such as Itgax 343 (CD11c), Spp1 and Clec7a 83,84 (Fig. 5a). Based on studies showing physiological upregulation of Mean expression in group Oasl2 Apoe     Fig. 3 c-d) Fig. 5b and Extended Data Fig. 9a).

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Therefore, the BM-derived MGLCs engrafted in the brain have a hybrid transcriptional identity co-352 expressing homeostatic adult microglia genes, BAM, and embryonic microglia genes.

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Genes primarily expressed in monocyte and dendritic cells 77, 86 were not detected in the brain cell 355 clusters, and no expression changes were found in MGLCs as compared to host and naïve MG 356 (Extended Fig. 9b . 5c-d). We also examined

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BMT/HSCT with a conditioning regimen that combines BU and CSF1Ri could be a promising 385 treatment for Progranulin (GRN) deficiency. This is because microglia and MGLCs express and 386 secrete high levels of GRN which can cross-correct other CNS cells (Fig. 6a) Fig. 6b-e). Consistent with this, analyses of GRN expression in the eye also showed 396 partial GRN restoration (24-32% of WT, Fig. 6f-h). in BMP levels in both Sham and Untreated Grn -/mice compared to WT (Fig. 6i-j). Most notably,

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suggesting that even partial reconstitution of GRN expression can restore lipid metabolism and 408 result in therapeutic benefit.

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However, compared to wild-type mice, T-cells and Ly6C+ had a higher fraction of host-derived 415 cells (71% ± 2.7 in SP and 65% in PB, Fig. 6m-n). As seen in the brain and eyes, BU + PLX +

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the inhibitor used, its receptor specificity, the total dose, and the treatment course may account 502 for discrepancies in toxicity highlighting the need to limit dose and exposure time.

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In addition to MG replacement, there is significant therapeutic potential for replacing tissue 505 macrophages (MF) and using these cells to deliver therapeutic proteins to tissues such as the 506 heart, lungs, and liver. In theory, a conditioning protocol that includes CSF1Ri could deplete tissue 507 MF and improve the replacement of these cells after transplantation. By investigating the 508 reconstitution of tissue MFs in various organs, we found that while the total engraftment of donor-

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Briefly, Kit+ cells were purified from total bone marrow (isolated as described above) using anti-    randomized. The Stranger mice were only exposed to one subject mouse per day.

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However, the nest has flat wall which is defined as less than 50% of the circumference of mouse 871 body height when curled up on its side. score of 5= More than 90% of the nestlet is torn and the 872 nest is a crater, with walls higher than 50% of the circumference of the mouse body height. If the 873 nesting score is ambiguous (e. g. a nest with a score of 5, but more than 10% of the nestlet un-874 shredded) a score of 4.5 was assigned.

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Brain and bone marrow cells were isolated, stained, and FAC-sorted as described above.

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(v/v/v) for lipid quantitation as described below.

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with an Ascentis Express guard holder that is connected to a 1290 LC system was used to separate lipids.

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The annotation MNH4+ represents measurement in positive mode where M indicates the neutral 997 mass while NH4+ is the ammonium adduct.

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Lipids were annotated and quantified using a high-throughput analysis software called

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MassHunter for qualitative analysis and QQQ quantitative analysis software known as Quant-My-